January 10, 2016

Central Dogma of T cell activation may be too simplistic

  • T cell activation is dependent on 3 signals
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Central Dogma of T cell activation may be too simplistic

  • The actuality is that the biology of GVHD is likely much more complex
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A Mixed Lymphocyte Reaction (MLR) is our best in vitro model of this complexity

  • PBMCS from two donors, irradiate one fraction and label other
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A Mixed Lymphocyte Reaction (MLR) is our best in vitro model of this complexity

  • PBMCS from two donors, irradiate one fraction and label other
  • Mix together
Your browser does not support SVG

A Mixed Lymphocyte Reaction (MLR) is our best in vitro model of this complexity

  • PBMCS from two donors, irradiate one fraction and label other
  • Mix together
  • Allow alloactivation of T cells to proceed over the following 3-5 days
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A Mixed Lymphocyte Reaction (MLR) is our best in vitro model of this complexity

  • PBMCS from two donors, irradiate one fraction and label other
  • Mix together
  • Allow alloactivation of T cells to proceed over the following 3-5 days
  • Dividing T cells will dilute out the green label (CFSE) and can be quantified flow cytometrically
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Primary experimental question

How can single cell RNA seq performed on cells from an MLR inform us of the transcriptional events that define alloproliferation


Overview of experiment

  • Three donors were utilized (A, B, C)
  • MLR was set up using each donor as a responder on each other donor (e.g A on B, A on C, B on A, B on C)
  • Controls included T cells cultured with anti-CD3/anti-CD28 microbeads
  • Cells were harvested and sorted by CFSE dilution (e.g. Division 0, Division 1, … Division 4)